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Ruchira Singh, M. Joseph Phillips, David Kuai, Jackelyn Meyer, Jessica M. Martin, Molly A. Smith, Enio T. Perez, Wei Shen, Kyle A. Wallace, Elizabeth E. Capowski, Lynda S. Wright, David M. Gamm; Functional Analysis of Serially Expanded Human iPS Cell-Derived RPE Cultures. Invest. Ophthalmol. Vis. Sci. 2013;54(10):6767-6778. doi: 10.1167/iovs.13-11943.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the effects of serial expansion on the cellular, molecular, and functional properties of human iPS cell (hiPSC)-derived RPE cultures.
Fibroblasts obtained from four individuals were reprogrammed into hiPSCs and differentiated to RPE cells using previously described methods. Patches of deeply pigmented hiPSC-RPE were dissected, dissociated, and grown in culture until they re-formed pigmented monolayers. Subsequent passages were obtained by repeated dissociation, expansion, and maturation of RPE into pigmented monolayers. Gene and protein expression profiles and morphological and functional characteristics of hiPSC-RPE at different passages were compared with each other and to human fetal RPE (hfRPE).
RPE from all four hiPSC lines could be expanded more than 1000-fold when serially passaged as pigmented monolayer cultures. Importantly, expansion of hiPSC-RPE monolayers over the first three passages (P1–P3) resulted in decreased expression of pluripotency and neuroretinal markers and maintenance of characteristic morphological features and gene and protein expression profiles. Furthermore, P1 to P3 hiPSC-RPE monolayers reliably demonstrated functional tight junctions, G-protein–coupled receptor-mediated calcium transients, phagocytosis and degradation of photoreceptor outer segments, and polarized secretion of biomolecules. In contrast, P4 hiPSC-RPE cells failed to form monolayers and possessed altered morphological and functional characteristics and gene expression levels.
Highly differentiated, pigmented hiPSC-RPE monolayers can undergo limited serial expansion while retaining key cytological and functional attributes. However, passaging hiPSC-RPE cultures beyond senescence leads to loss of such features. Our findings support limited, controlled passaging of patient-specific hiPSC-RPE to procure cells needed for in vitro disease modeling, drug screening, and cellular transplantation.
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